Nitroso formyl tetrahydropteroic amino acids thereof



Patented Mar. 10, 1953 UNITED STATE s PATENT orn-ca assust NITRO'SO FO-RMY L TETRAHXDROPTEROIG AMINO (AGIDS THEREOF 5 Claims. 1

This invention relates to new "organic "compounds having biological activity. More particularly, it relates to nitroso forrnyl tetrahydropteroylglutamic "acid and related compounds and methods of preparation thereof.

In my application, Serial Number 102,947',"now U. S. Patent 2,537,006, I reviewed the effect of nitrous .acid on 2-amino-4-hydroxy .pteridines as shown in the prior art. The prior art indicates that when nitrous acid is reacted with '2- amino-4-hydroxy pteridines the following results take place: (1.) destruction of the ring system, (2) desamination of the 'Z-am'ino group, (3) simultaneous desamination in the '2-ip'osition and nitrosation of the ill-nitrogen atom of pteroic acid, (4) biological inactivation of folic acid. The prior :art further shows that the reactions described above were c'arried out in an excess of nitrous acid at temperatures ranging from room temperature to 100 C.

In a pending application of my coworkers Brockma'n and Roth, Serial Number 153,294, filed March 31, 1950, there is described a method of preparing compounds having citrovorum factor activity. These compounds are prepared by reducing formyl pteroylglutamic acid and related compounds to obtain the corresponding formyl tetrahydro -pteroylglutamicacidand related compounds.

I have now found that formyl tetrahydropteroic acid "and amino acid amides thereof can be nitrosated under the conditions described hereinafter to produce nitroso derivatives of formyl tetrahydropteroic acid and amino acd amides thereof. 'It is entirely unexpected that these compounds are active for Leu'conostoc citrovorum, or in other words, have citrovorum factor activity since it has been reported previously (Journal of Biological Chemistry 185, 405 (1950)) that'the citrovorum factor loses its activity in aqueous solution at pH 2. Shive-reported in his paper Synthetic Members of the Fol inic Group (J. A. C. S. '72, 2818, June 1950-): The activity of the synthetic material derived from folic acid is destroyed by dilute nitrous acid. Since nitrousacidis also an oxidizing agent and it is reacted with products in a reduced state, it could be not foreseen that the nitrous acid would not oxidize the formyl tetrahydropteroylglutamic acid or related compounds and thus inactivate them. The exact structure of the new compounds produced by the nitrosation of formyl tetrahydropteroic acid and amino acid amides thereof has not been definitely determined as yet due to their complex nature. It is believed, however, that they may be represented by one of the following formulae:

In these, R represents a hydroxyl or amino acid radical. It will be understood that both of the above may exist in tautomeric forms depending upon the "conditions in which they are present.

The compounds of the present invention are prepared by reacting a formyl tetrahydropteroic acid or amino acid amide thereof with nitrous acid at an acid pH of 2 or lower. It is preferred that the nitrous acidbe prepared in situ from a nitrite salt, such as an alkali metal nitrite, and an acid since nitrousacid itself is somewhat onstable and difficult to handle.

The intermediates used in the reaction o'f'the present invention can be prepared by the rei duction of formyl pteroic acid and amino acid amides thereof with hydrogen and a noble metal or by the formyl'ation of tetrahydropteroic acid or amino acid amides thereof. The preferred intermediate is formyl tetrahydropteroylglutamic acid, and preferably 5-fo'rmyl tetrahydropter'oylglutamic acid, however, other amino acid amides o'f .pteroic acid can be used such as glycine, as- ;partic acid, leucine, serine, sarcosine, phenylalanine, alanine, isovaline, cystine, and the like.-

The "reaction "of the present invention will "take .place'overa wide range of acidity from about pH -2 down to the acidity of strong mineral acid. However, it is important. that the nitrosationbe carried out immediately after the starting material is placed in contact with the acid medium to avoid any isomerization of the starting product. I prefer to use a mineral acid such as hydrochloric or sulfuric at such a concentration that the intermediates will be in solution at about 0 C. The reaction is carried out by adding to the acidic solution of the intermediate 2. molecular equivalent of nitrous acid.

It is desirable to carry out the reaction at a temperature within the range of from 10 C. to +10 C. and preferably from about 5 C. to +5 C.

The reaction is usually completed in a matter of minutes and the end of the reaction may be determined by a test with starch iodide paper or paste in a manner familiar to those skilled in the art. In general, the nitroso formyl tetrahydropteroic acid or amino acid amide thereof precipitates from the cold solution by adjustment of the pH to 2-4 and it can be conveniently separated by filtration.

Some of the compounds of the present invention, such as nitroso formyl tetrahydropteroylglutamic acid, are useful as growth promoters of various organisms and also in the reversal of toxicity of aminopterin and other derivatives of pteroylglutamic acid which are metabolite antagonists. These properties make them of potential importance as drugs particularly in the therapy of certain leukemias.

The process and representative compounds of the invention will now be described in detail in the following examples. All parts are by weight unless otherwise indicated.

Example 1 adjusted to pH 3-4'by the addition of dilute sodium hydroxide, whereupon a granular creamcolored precipitate appears. After filtering, Washing and drying, 0.422 part of the nitroso derivative of formyl tetrahydropteroylglutamic acid is obtained. Analysis shows the presence of one nitroso group.

Example 2 About 185 parts by volume of 1.0 N hydrochloric acid is cooled to -5 C. Then 2.5 parts of the barium saltof formyl tetrahydropteroylglutamic acid (1.375 parts of free citrovorum factor) is'added and immediately the addition of 0.202 part Of sodium nitrite (dissolved in 5 parts of water) is begun. During the addition complete solution results. While the solution is cooled, dilute sodium hydroxide is added to pH 2.5-3.5. After allowing to cool for some time, the cream-colored precipitate is filtered, washed and dried to yield 0.8615 part of the nitroso -derivative of formyl tetrahydropteroylglutamic acid, which can be purified by recrystallization from water.

This compound melts with decomposition at 240-245 0.; gives a positive Liebermann nitroso test and has, in 0.1 N sodium hydroxide at a concentration of Ihg/liter, one maximum at 275 m When measured as a growth factor for the organism Leuconostoc citrovorum it was .found to be active. In'the reversal of aminopterin it was found to be about three times as active as pteroylglutamic acid or its nitroso derivative.

Microanalysis of this new product gives the values calculated for the dihydrate of nitroso formyl tetrahydropteroylglutamic acid.

I claim:

1. N- (4-[2-amino-4-hydroxy-5-formyl-5,6.7,8- tetrahydropyrimido [4,5-b] pyrazyl-G-methyl-N- nitrosoaminolbenzoyl) glutamic acid.

2. A method which comprises mixing together in an acid aqueous solution having a pH less than 2, at a temperature within the range of -10 C. to +10 C. N-(4-[2-amino-4-hydroxy-5-formyl- 5,6,7,8 tetrahydropyrimido [4,5 b] pyrazyl 6- methylaminolbenzoyl) glutamic acid and nitrous acid and after reaction thereof recovering N-(4- [2 amino-4-hydroxy-5-formy1 5,6,7,8 tetrahydropyrimido[4,5 blpyrazyl 6 methyl-N-nitrosoamina] benzoyl) glutamic acid.

3. A compound having the formula:

COOH

t H t N I Iom-NQg-rm- E 0 NHz-L H N N H H (g OOH in which X and Y are members of the group consisting of formyl and nitroso radicals, one of which is a formyl radical, and R is an integer of 1 to 3, inclusive.

4. A compound having the formula:

in which R is an integer of 1 to 3, inclusive.

5. A method which comprises reacting together a compound having the formula:

in which R is an integer of 1 to 3, inclusive, with nitrous acid in a substantially aqueous solvent at a temperature within the range of -10 C. to +l0 C. and after reaction thereof recovering a compound having the formula:

HO=O OH I N=O N H I o coon OHg-NQ-Nliifl l l Hon \IH 11 5 N g H 0011 in which R is as defined above.

DONNA B. COSULICH.

No references cited. 

3. A COMPOUND HAVING THE FORMULA:
 5. A METHOD WHICH COMPRISES REACTING TOGETHER A COMPOUND HAVING THE FORMULA: 